CD25 KO mice undergo normal B cell development and maturation. (A) Absolute number of live cells in bone marrow (BM) and spleen. (B) Frequency of B220⁺ B cells among live singlets in BM and spleen. (C) Gating strategy for and frequency of pro-B & pre-B, immature and mature recirculating B cells in the BM. Gated on B220⁺ live singlets. (D) Representative gating strategy and frequencies of pre-B (CD43^-) and pro-B (CD43⁺) cells in the BM. Gated on IgD^-IgM^-B220⁺ live singlets. (E) Volcano plot of pre-B cell RNA sequencing data showing log₂ fold change on the x axis and -log₁₀ (P-value) on the y axis. Top 3 differentially expressed transcripts are depicted by purple dots (false discovery rate [FDR] < 0.05, |log₂ fold change| > 1, transcripts per million [TPM] > 10). Dotted lines indicate p-value = 0.05 and log₂ fold change = ±1. (F) Analysis of reactivity of IgG and IgM serum antibodies in CD25-KO and WT mice with a 128-autoantigen microarray (n = 4 pairs of 3 to 4-month-old CD25-KO and CD25-WT littermates). The data are plotted as a volcano plot with log₂ fold change on the x axis and -log₁₀ (P-value) on the y axis. (G) Gating strategy for follicular and T2 transitional splenic B cells (gated on live B220⁺CD23⁺ singlets), and marginal zone and T1 transitional splenic B cells (gated on live B220⁺CD23^- singlets). (H) Quantifications of the frequency of the splenic mature B cell subsets from Fig. 1G across experimental groups. Data are representative of 3 independent experiments (BM) or 5 independent experiments (spleen) with 2 to 4 mice per genotype (A–D, G, H). Error bars represent mean ± std. dev.